腺病毒7型河南分离株全基因序列测定及重组分析

目的了解腺病毒7型河南分离株（hAdV35/Henan/2010）全基因特征及与其他腺病毒之间的进化重组关系。方法设计39对全长引物,采用RT-PCR方法扩增河南分离株腺病毒全序列,用DNASTAR中的SeqMan拼接全序,运用BioEdit进行序列剪齐,运用Mega4.0分析基因组编码蛋白hexon、fiber、E4和penton区域氨基酸与核苷酸的同源性,并与其他腺病毒序列绘制进化树,运用Simplot软件进行序列重组分析。结果 hAdV35/Henan/2010基因组全长35 234bp,与其他腺病毒相比,基因组氨基酸和核苷酸同源性为68.0%-99.8%和46.5%-99.5%。不同编码区hexon、fiber、E4和penton的核苷酸同源性分别为72.4%-95.4%、74.1%-97.0%、55.7%-92.7%和69.9%-99.3%。进化树分析显示河南分离株与腺病毒7型疫苗株同属一支,与腺病毒3型进化分支最近,Bootsacn分析显示与B亚属腺病毒在多个区域发生重组,尤其与腺病毒3在hexon区域高度重组。结论 hAdV35/Henan/2010与B族腺病毒亲缘关系较近,与hAdV3在hexon区域存在重组。     

Structure-function analysis of pneumococcal DprA protein reveals that dimerization is crucial for loading RecA recombinase onto DNA during transformation

Transformation promotes genome plasticity in bacteria via RecA-driven homologous recombination. In the Gram-positive human pathogen Streptococcus pneumoniae, the transformasome a multiprotein complex, internalizes, protects, and processes transforming DNA to generate chromosomal recombinants. Double-stranded DNA is internalized as single strands, onto which the transformation-dedicated DNA processing protein A (DprA) ensures the loading of RecA to form presynaptic filaments. We report that the structure of DprA consists of the association of a sterile alpha motif domain and a Rossmann fold and that DprA forms tail-to-tail dimers. The isolation of DprA self-interaction mutants revealed that dimerization is crucial for the formation of nucleocomplexes in vitro and for genetic transformation. Residues important for DprA-RecA interaction also were identified and mutated, establishing this interaction as equally important for transformation. Positioning of key interaction residues on the DprA structure revealed an overlap of DprA-DprA and DprA-RecA interaction surfaces. We propose a model in which RecA interaction promotes rearrangement or disruption of the DprA dimer, enabling the subsequent nucleation of RecA and its polymerization onto ssDNA.     

Microbial genome dynamics in CNS pathogenesis

The balancing act between microbes and their host in commensal and disease states needs to be deciphered in order to fully treat and combat infectious diseases. The elucidation of microbial genome dynamics in each instance is therefore required. In this context, the major bacterial meningitis pathogens are Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae. In prokaryotic CNS pathogenesis both the intact organism as well as its released components can elicit disease, often resulting in neurological sequelae, neurodegeneration or fatal outcome. The study of microbial virulence in CNS disease is expected to generate findings that yield new information on the general mechanisms of brain edema and excitatory neuronal disturbances due to meningitis, with significant potential for discoveries that can directly influence and inspire new strategies for prevention and treatment of this serious disease.     

Host transcription factor Rpb11p affects tombusvirus replication and recombination via regulating the accumulation of viral replication proteins

Previous genome-wide screens identified over 100 host genes whose deletion/down-regulation affected tombusvirus replication and 32 host genes that affected tombusvirus RNA recombination in yeast, a model host for replication of Tomato bushy stunt virus (TBSV). Down-regulation of several of the identified host genes affected the accumulation levels of p33 and p92(pol) replication proteins, raising the possibility that these host factors could be involved in the regulation of the amount of viral replication proteins and, thus, they are indirectly involved in TBSV replication and recombination. To test this model, we developed a tightly regulated expression system for recombinant p33 and p92(pol) replication proteins in yeast. We demonstrate that high accumulation level of p33 facilitated efficient viral RNA replication, while the effect of p33 level on RNA recombination was less pronounced. On the other hand, high level of p92(pol) accumulation promoted TBSV RNA recombination more efficiently than RNA replication. As predicted, Rpb11p, which is part of the polII complex, affected the accumulation levels of p33 and p92(pol) as well as altered RNA replication and recombination. An in vitro assay with the tombusvirus replicase further supported that Rpb11p affects TBSV replication and recombination only indirectly, via regulating p33 and p92(pol) levels. In contrast, the mechanism by which Rpt4p endopeptidase/ATPase and Mps1p threonine/tyrosine kinase affect TBSV recombination is different from that proposed for Rpb11p. We propose a model that the concentration (molecular crowding) of replication proteins within the viral replicase is a factor affecting viral replication and recombination.     

Activation-induced cytidine deaminase induces DNA break repair events more frequently in the Ig switch region than other sites in the mammalian genome

Activation-induced cytidine deaminase (AID) produces DNA breaks in immunoglobulin genes during antibody diversification. Double-stranded breaks (DSB) in the switch region mediate class switch recombination, and contribute to gene conversion and somatic hypermutation in the variable regions. However, the relative extent to which AID induces DSB in these regions or between these and other actively expressed sequences is unknown. Here, we exploited an enhancer-trap plasmid that identifies DSB in actively expressed loci to investigate the frequency and position of AID-induced vector integration events in mouse hybridoma cells. Compared to control cells, wild-type AID stimulates plasmid integration into the genome by as much as 29-fold. Southern and digestion-circularization PCR analysis revealed non-uniformity in the integration sites, with biases of 30- and 116-fold for the immunoglobulin kappa light chain and mu heavy chain genes, respectively. Further, within the immunoglobulin mu gene, 73% of vector integrations map to the mu switch region, an enhancement of five- and 12-fold compared to the adjacent heavy chain variable and mu gene constant regions, respectively. Thus, among potential highly transcribed genes in mouse hybridoma cells, the immunoglobulin heavy and light chain genes are important AID targets, with the immunoglobulin mu switch region being preferred compared to other genomic sites.     

PARP inhibitors for homologous recombination-deficient prostate cancer

Introduction: Prostate adenocarcinoma represents a leading cause of cancer-related mortality. Increased emphasis on understanding the molecular basis of prostate cancer has identified a substantial burden of homologous recombination (HR) pathway mutations, which are enriched in castrate-resistant disease. This discovery has yielded novel therapeutic opportunities.

Areas covered: We will discuss the treatment of castrate-resistant prostate cancer (CRPC), with a focus on the use of poly (ADP-ribose) polymerase (PARP) inhibitors in this space. Evidence for use in HR-deficient patients will be outlined with discussion of the mechanism of action for this drug class, pathways of resistance, and approaches for expanding PARP inhibitor use to non–HR-deficient prostate cancer subgroups.

Expert opinion: PARP inhibition represents an exciting tool for management of HR-inactivated CRPC. With rapid adoption of next-generation sequencing technologies and other molecular techniques, the number of patients in this category is likely to increase. Ongoing and future investigations will be critical for improved understanding of the promise and appropriate treatment sequencing of PARP inhibition and optimal options for HR-proficient and -deficient prostate cancer populations. Questions remain about the clinical significance of monoallelic vs. biallelic HR mutations, the relevance of germline vs. somatic-only mutations, and the importance of mutations in non-canonical HR genes.     

Origin and epidemic status of porcine epidemic diarrhea virus variants in China

From 2010, porcine epidemic diarrhea virus (PEDV) variants caused sequential outbreaks of disease in Asia and the United States. In this retrospective study, 49 complete spike (S) gene sequences were obtained from PEDV strains collected in China from 2014 to 2016. We observed that variant PEDV strains with novel insertions, deletions, and multiple S gene recombination types were present in China. In addition, mixed infections involving different variant strains were observed in some areas. Based on phylogenetic and recombination analyses, we determined that the newly emerged PEDV variants potentially originated via recombination between the earliest Chinese G1 genogroup strain, JS‐2004‐2 and earlier Korean pandemic strains. These findings provide important information for understanding ongoing PEDV outbreaks and suggest that novel variants make it more difficult to prevent PEDV infection.     

A recombination between two Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV‐1) vaccine strains has caused severe outbreaks in Danish pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) is prevalent in Danish swine herds. In July 2019, PRRSV‐1 was detected in a PRRSV‐negative boar station and subsequently spread to more than 38 herds that had received semen from the boar station. Full genome sequencing revealed a sequence of 15.098 nucleotides. Phylogenetic analyses showed that the strain was a recombination between the Amervac strain (Unistrain PRRS vaccine; Hipra) and the 96V198 strain (Suvaxyn PRRS; Zoetis AH). The major parent was the 96V198 strain that spanned ORFs 1–2 and part of ORF 3 and the minor parent was the Amervac strain, which constituted the remaining part of the genome. The virus seems to be highly transmissible and has caused severe disease in infected herds despite a high level of genetic identity to the attenuated parent strains. The source of infection was presumable a neighbouring farm situated 5.8 km from the boar station.